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Typescript (photocopy). / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1978 G37 / Master of Science

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The in vitro study of muscle cell growth is hampered by the presence of non-muscle cells, particularly fibroblasts. The heterobifunctional cross-linking agent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) has been used to create a novel "toxic growth factor" to address the problem. Epidermal growth factor (EGF), which stimulates fibroblast but not satellite cell proliferation, was conjugated via SPDP to a potent ribosome inhibitor, pokeweed antiviral protein (PAP). By preferentially binding to fibroblasts, it was hoped that EGF-PAP could cytotoxically eliminate fibroblasts from primary cultures of rat skeletal muscle satellite cells. While EGF-PAP did prove to be a fibroblast cytotoxin, it could not completely eliminate them from cell cultures. Low dose-time exposures improved the ratio of multinucleated cells to mononucleated cells (percent fusion) by up to 66% over controls, but increased concentrations, or durations of EGF-PAP treatment, proved detrimental to satellite cell growth and/or differentiation.

A polpa dental é formada de tecido conjuntivo frouxo sendo constituída por diversas células, dentre as quais os fibroblastos são as mais numerosas. Ao serem submetidas a agressões diversas, estas células respondem com a liberação de substâncias, tais como citocinas e quimiocinas, que participam de maneira ativa no processo inflamatório. Assim sendo, este trabalho teve como proposição: 1. avaliar a capacidade de fibroblastos de polpa dental humana em cultura em produzirem as quimiocinas MIP-l\'alfa\' /CCL3 e SDF-1/CXCL12; 2. avaliar a produção destas quimiocinas pelos fibroblastos quando estimulados por Enterococcus faecalis morto por calor com relação à quantidade de bactérias por célula e 3. avaliar a liberação destas quimiocinas com relação ao tempo de estímulo. Para o estabelecimento das culturas, foi coletada a polpa de terceiro molar hígido de um paciente saudável. O tecido foi extraído, armazenado e picotado em meio de cultura para fibroblastos (DMEM), os quais foram utilizados a partir da quarta passagem. Após adesão das células a placas de 24 poços, o meio de cultura contendo Enterococcus .faecalis morto por calor numa concentração correspondente a 1, 10 e 100 bactérias por fibroblasto foi adicionado aos poços. Após 1, 6 e 24 horas, o sobrenadante das células foi coletado para a análise por ELISA. A análise estatística foi realizada aplicando-se o teste Kruskal-Wallis com nível de significância de 5%. A produção de MIP-l\'alfa\' /CCL3 e SDF-l/CXCL12 pelas células pôde ser detectada por ELISA. Os fibroblastos pulpares se mostraram capazes de produzir SDF-1 constitutivamente sendo que o estímulo bacteriano levou a uma diminuição estatisticamente significativa desta produção. A produção de MIP-l\'alfa\' também foi detectada tanto de maneira constitutiva como em resposta ao desafio microbiano. Enquanto a concentração intermediária de bactéria por fibroblasto (10:1) mostrou uma produção semelhante ao grupo controle, as concentrações de 1 e 100 bactérias por fibroblasto induziram aumento maior na primeira hora de estímulo. Essas diferenças, entretanto, não foram estatisticamente significativas. A capacidade dos fibroblastos secretarem quimiocinas, como MIP-l\'alfa\' e SDF-1, reforça a importância dessas células dentro do contexto de imunidade e inflamação pulpar, principalmente por serem as células mais numerosas deste microambiente. / Dental pulp is a connective tissue structure constituted by many different cell types. Among them, the fibroblasts are the most frequent ones. When challenged by different aggressive agents, these cells are able to release some substances like cytokines and chemokines, which are essential to trigger the inflammatory process. The aims of this study were: 1. to evaluate the ability of fibroblasts to produce the chemokines MIP-l\'alfa\'/CCL3) and SDF-1/CXCL12; 2. to evaluate the expression of these chemokines by fibroblasts when challenged by heat killed Enterococcus. faecalis in gradual concentrations and 3. to evaluate the production of these chemokines in a time course manner. The dental pulp from non-carious third molar was collected from a healthy patient. Explants were made and stocked in culture medium (DMEM) for fibroblasts growth. The cells were used since passage four. In a 24-well plate and after reaching confluence, culture medium alone or containing heat killed E. faecalis at proportion 1:1, 10:1 and 100:1 bacteria:fibroblast, were added to the fibroblasts. After 1, 6 and 24 hours, the supernatants were collected for analysis. The protein detection of MIP-l\'alfa\'/CCL3 and SDF-1/CXCL12 was performed by ELISA. For statistical analysis, data were assessed by Kruskal-Wallis followed by Miller post-test. Significance levels of 5% were adopted. Production of both chemokines was detected by ELISA. Pulp fibroblasts were able to produce SDF-1 constitutively. This production decreased with the increase in the number of heat killed E. faecalis increased (p < 0.05). Production of MIP-l\'alfa\' was detected in unchallenged and challenged cells. The median bacterial concentration (10:1) presented a profile production similar to that of unstimulated cells. Bacterial concentrations of 1 and 100 microrganisms/cell showed a highly enhanced production of MIP-l\'alfa\' at the first hour of stimulum; however, these data were not statistically significant (p > 0.05). Fibroblasts ability to produce chemokines, like MIP-l\'alfa\' and SDF-1, confirms their importance at immune and inflammatory events in dental pulp, specially being fibroblasts the most abundant cells at this microenvironment .

Zhang, Yang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 179-201). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Telomerase is an enzyme that helps maintain the telomeric ends of chromosomes during DNA replication. Telomere lengths represent a balance between telomerase activity attempting to elongate their ends, and cell division that causes telomere shortening. As cells age, diminished telomerase activity allows a shortening of telomere lengths until they reach a target length that stimlulates apoptosis. Identifying a drug capable of upregulating telomerase activity may help increase cell (and even organismal) lifespan. The purpose of this thesis was to determine whether treatment of human primary foreskin fibroblast cultures with a 14 amino acid (aa) ependymin peptide mimetic upregulates (or at least maintains) telomerase activity and telomere lengths during cellular ageing. The 14aa peptide was previously shown to significantly increase the murine lifespan by 25%, so its activity was a logical candidate to test in this thesis. In a preliminary set of experiments, the human primary fibroblast cells were shown to respond to the 14aa drug by upregulating the antioxidative enzyme superoxide dismutase (SOD), thus human fibroblast cells likely contain the appropriate receptor for binding this drug. This same dose proved optimal for upregulating telomerase activity in the fibroblast cells an average of 57% relative to untreated cells (p value = 0.003). The upregulation appears to be specific for the sequence of aa in the 14aa drug since a“scrambled" peptide containing the same aa but in a different order showed no upregulation, even at doses 10-fold higher. Treatment of mice once per day or twice per day with the 14aa peptide was also found to upregulate telomerase activity in vivo in brain and heart. The activity was optimal at a 3.3 mg/kg dose for each aged organ, and was generally high in young organs. The activity observed in heart was a total surprise since heart cells are generally thought to be quiescent, and telomerase is usually associated with cell division, so perhaps telomerase has a function other than in cell division. The second part of the hypothesis tested whether treatment of fibroblast cells with the 14aa drug elongated (or prevented from shortening) telomere lengths in aged cells. A telomere length assay (TLA) based on a Southern hybridization approach using a telomere probe appeared to work well, since marker DNAs showed appropriate differences in their“telomere smears", and aged fibroblast cells showed shorter smears than young cells. However, no difference was observed between drug-treated versus vehicle-treated cells, even at the 10 ng/ml dose previously shown to strongly upregulate telomerase activity. So perhaps the upregulation of telomerase activity was not sufficient to provide a measurable increase in telomere lengths. Telomerase has been shown to extend the lifespan of virus-transformed human cells without showing any visible telomere lengthening (Blackburn et al, 1999), so perhaps telomerase can increase cell lifespan without increasing telomere lengths. To our knowledge, this is the only drug demonstrated to upregulate telomerase activity. Transforming cells with the viral T-antigen can upregulate telomerase, but T-antigen is not a therapeutic drug since it also causes cancer. Telomerase upregulation is known to occur during oncogenesis, but telomerase itself is not an oncogene since oncogenesis also requires the upregulation of oncogenes. Our lab previously showed this peptide does not upregulate the potent oncogene myc. If this proves to be the case for other oncogenes, using this 14aa drug to upregulate telomerase activity without activating oncogenes could prove extremely useful for helping prove telomerase is not an oncogene, and for extending cell lifespan.